Polypeptide analogues having growth hormone releasing activity

ABSTRACT

Novel peptides of the formula A 1  -A 2  -Ala-Trp-DPhe-A 5  are disclosed which promote the release of growth hormone when administered to animals. These peptides can be used therapeutically.

This invention relates to novel polypeptide compounds which promote therelease of growth hormone when administered to animals, preferablyhumans. In another aspect, this invention relates to methods forpromoting the release and elevation of growth hormone levels in animalsby administration of specified growth hormone releasing polypeptidecompounds thereto.

BACKGROUND OF THE INVENTION

The elevation of growth hormone (GH) levels in animals, e.g., mammalsincluding humans, upon administration of GH-releasing compounds can leadto enhanced body weight and to enhanced milk production if sufficientlyelevated GH levels occur upon administration. Further, it is known thatthe elevation of growth hormone levels in mammals and humans can beaccomplished by application of known growth hormone releasing agents,such as the naturally occurring growth hormone releasing hormones.

The elevation of growth hormone levels in mammals can also beaccomplished by application of growth hormone releasing peptides, someof which have been previously described, for example, in U.S. Pat. No.4,223,019, U.S. Pat. No. 4,223,020, U.S. Pat. No. 4,223,021, U.S. Pat.No. 4,224,316, U.S. Pat. No. 4,226,857, U.S. Pat. No. 4,228,155, U.S.Pat. No. 4,228,156, U.S. Pat. No. 4,228,157, U.S. Pat. No. 4,228,158,U.S. Pat. No. 4,410,512, U.S. Pat. No. 4,410,513.

Antibodies to the endogenous growth hormone release inhibitor,somatostatin (SRIF) have also been used to cause elevated GH levels. Inthis latter example, growth hormone levels are elevated by removing theendogenous GH-release inhibitor (SRIF) before it reaches the pituitary,where it inhibits the release of GH.

Each of these methods for promoting the elevation of growth hormonelevels involve materials which are expensive to synthesize and/orisolate in sufficient purity for administration to a target animal.Short chain, low molecular weight, relatively simple polypeptides whichare relatively inexpensive to prepare and have the ability to promotethe release of growth hormone would be desirable because they should bereadily and inexpensively prepared, easily modified chemically and/orphysically, as well as easily purified and formulated; and they shouldhave excellent transport properties.

Although some short chain polypeptides which can promote the release andelevation of growth hormone levels in the blood are known, it isimportant to be able to tailor polypeptides for a variety of reasons,such as delivery, bioabsorbance, increased retention time, etc. However,amino acid changes at certain positions can have dramatic effects on theability of short chain peptide to promote the release of a growthhormone.

It would be desirable to have different short chain polypeptides whichcan promote the release and elevation of growth hormone levels in theblood of animals, particularly in humans. It would also be useful to beable to use such polypeptides to promote the release and/or elevation ofgrowth hormone levels in the blood of animals and humans.

It would also be desirable to provide methods for promoting the releaseand/or elevation of growth hormone levels in the blood of animals usingsuch short chain polypeptides.

SUMMARY OF THE INVENTION

The polypeptides are defined by the following formula:

A₁ -A₂ -Ala-Trp-DPhe-A₅, where

A₁ is Gly, DAla, β-Ala, His, Ser, Met, Pro, Sar, Ava, Aib, a N-loweralkylaminocarboxylic acid, a N,N-bis-lower alkyl amino-carboxylic acidor a lower alkyl aminocarboxylic acid, wherein the lower alkyl groupcomprises 2 to about 10 straight-chain carbon atoms. A₁ is preferablyDAla.

A₂ is DTrp,D.sup.β Nal, D-4-Y-Phe-or 5-Y-D-Trp, wherein Y is OH, Cl, Br,F or H. More preferably, A₂ is D.sup.β Nal.

A₅ is A₃ -A₄ -A_(5'), A₃ -A_(5'), A₄ -A_(5') or A_(5'). Preferably A₅ isA_(5'). A₃ is Ala, Gly, DAla, Pro or desAla. A₄ is Ala, Gly, DAla, Pro,a linear lower alkyl aminocaboxylic acid, or desAla, A_(5') is Lys(ξ-R₁,R₂)-Z, Orn(δ-R₁, R₂)-Z, NH(CH₂)_(x) N(R₃, R₄). A₅, can also be Lys-Z,Orn-Z or Arg-Z when A₁ is not His. R₁ is a linear lower alkyl group or Hatom. R₂ is a linear lower alkyl group or H atom. When R₁ is H, R₂ isnot H; similarly when R₂ is H, R₁ is not H. R₃ is a linear lower alkylgroups or H atom. R₄ is a linear lower alkyl group or H atom. The loweralkyl group comprises 2 to about 10 straight-chain carbon atoms. Z isNH(linear lower alkyl groups, N(linear lower alkyl group)₂, O-(linearlower alkyl group), NH₂ or OH, wherein the linear lower alkyl group isas defined above. x is 2 through 15. And organic or inorganic additionsalts of any of said polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered several novel short chain polypeptides which promotethe release and elevation of growth hormone levels in the blood ofanimals. The polypeptides are defined by the following formula:

A₁ -A₂ -Ala-Trp-DPhe-A₅, where

A₁ is Gly, DAla, β-Ala, His, Ser, Met, Pro, Sar, Ava, Aib, imidazoleacetic acid, a N-lower alkyl aminocarboxylic acid, a N,N-bis-lower alkylamino-carboxylic acid or a lower alkyl aminocarboxylic acid, wherein thelower alkyl group comprises 2 to about 10 straight-chain carbon atoms.Preferably, the lower alkyl group is 2 to 6 straight-chain carbon atoms.The lower alkyl group can be substituted or unsubstituted. Preferredsubstituents are O, N or Si. Preferably, the lower alkyl group isunsubstituted. Preferably, A₁ is Gly, DAla or His. Still more preferablyA₁ is His or DAla. A₁ is most preferably DAla.

A₂ is DTrp,D.sup.β Nal, D-4-Y-Phe-or 5-Y-D-Trp, wherein Y is OH, Cl, Br,F or H. Preferably, A₂ is D.sup.β Nal, D-4-Y-Phe or 5-Y-D-Trp. Morepreferably, A₂ is D.sup.β Nal. Y is preferably OH or H.

A₅ is A₃ -A₄ -A_(5'), A₃ -A_(5'), A₄ -A₅, or A_(5'). Preferably A₅ isA_(5'). A₃ is Ala, Gly, DAla, Pro or desAla. A₄ is Ala, Gly, DAla, Pro,a linear lower alkyl aminocaboxylic acid, or desAla, A₅, isLys(ξ-R₁,R₂)-Z, Orn(δ-R₁, R₂)-Z, LArg (g-R₅ -R₆) NH(CH₂)_(x) N(R₃, R₄).A_(5') can also be Lys-Z, Orn-Z or Arg-Z when A₁ is not His. R₁ is alinear lower alkyl group or H atom. R₂ is a linear lower alkyl group orH atom. When R₁ is H, R₂ is not H; similarly when R₂ is H, R₁ is not H.R₃ is a linear lower alkyl groups or H atom. R₄ is a linear lower alkylgroup or H atom. R₅ and R₆ are linear lower alkyl groups. The loweralkyl group comprises 2 to about 10 straight-chain carbon atoms.Preferably, the lower alkyl group is 2 to 6 straight-chain carbon atoms.The lower alkyl group can be substituted or unsubstituted. Preferredsubstituents can be O, N or Si. Preferably, the lower alkyl group isunsubstituted. g is guanidino. Z is NH(linear lower alkyl group),N(linear lower alkyl group)₂, O-(linear lower alkyl group), NH₂ or OH,wherein the linear lower alkyl group is as defined above. x is 2 through15. x is preferably 2 through 6. A₅, is preferably Lys-NH₂. And organicor inorganic addition salts of any of said polypeptides.

The amino acid residue abbreviations used are in accordance withstandard peptide nomenclature:

Gly=Glycine

Tyr=L-Tyrosine

Ile=L-Isoleucine

Glu=L-Glutamic Acid

Thr=L-Threonine

Phe=L-Phenylalanine

Ala=L-Alanine

Lys=L-Lysine

Asp=L-Aspartic Acid

Cys=L-Cysteine

Arg=L-Arginine

Ava=Aminovaleric acid

Aib=Aminoisobutyric acid

Gln=L-Glutamine

Pro=L-Proline

Leu=L-Leucine

Met=L-Methionine

Ser=L-Serine

Asn=L-Asparagine

His=L-Histidine

Trp=L-Tryptophan

Val=L-Valine

DOPA=3,4-Dihydroxyphenylalanine

Met(O)=Methionine Sulfoxide

Abu=α-Aminobutyric Acid

iLys=N.sup.ε -Isopropyl-L-Lysine

4-Abu=4-Aminobutyric Acid

Orn=L-Ornithine

D.sup.α Nal=α-Naphthyl-D-Alanine

D.sup.β Nal=β-Naphthyl-D-Alanine

Sar=Sarcosine

LArg=homoArgninine

All three letter amino acid abbreviations preceded by a "D" indicatedthe D-configuration of the amino acid residue, and glycine is consideredto be included in the term "naturally occurring L-amino acids."

The flexibility associated with the choice of basic, neutral or acidicamino acid residues that can be used for amino acids A₁, A₂, A₃ A₄ andA₅ provides a great deal of control over the physiochemical propertiesof the desired peptide. Such flexibility provides important advantagesfor the formulation and delivery of the desired peptide to any givenspecies. Also, these changes may improve oral absorption as well asmetabolism and excretion of the peptides. The moieties of R₁, R₂, R₃, R₄and Z can be varied as well, thereby providing added control over thephysiochemical properties of the desired compound. Consequently, one canobtain enhanced delivery of a peptide to a particular receptor and inparticular species.

Preferred growth hormone releasing compounds employed in the practice ofthe present invention are:

A₁ -A₂ -Ala-Trp-DPhe-A₅,

or organic or inorganic addition salts of any polypeptides; where A₁, A₂and A₅, are as defined above.

In a preferred embodiment, the growth hormone releasing peptide employedin the practice of the present invention has the formula:

DAla-A₂ -Ala-Trp-DPhe-A₅,

and organic or inorganic addition salts thereof. Preferred members ofthis group of compounds have the formula:

DAla-D.sup.β Nal-Ala-Trp-DPhe-LysNH₂

DAla-D.sup.β Nal-Ala-Trp-DPhe-Lys(ε-iPr)NH₂

DAla-DTrp-Ala-Trp-DPhe-LysNH₂

DAla-D.sup.β Nal-Ala-Trp-DPhe-NH(CH₂)₅ NH₂, and

NH₂ (CH₂)₅ CO-D.sup.β -Nal-Ala-Trp-D-Phe-NH(CH₂)₅ NH₂

as well as organic or inorganic addition salts thereof.

These compounds are presently the most preferred because these shorterchain polypeptides are less expensive to synthesize, and these specificcompounds have been shown to have a high level of potency at promotingthe increase in serum growth hormone levels.

These compounds are typically easy to synthesize, have efficacy atpromoting an increase in serum growth hormone levels, and are desirablefor commercial scale production and utilization. In addition, thesecompounds maybe advantageous in having physiochemical properties whichare desirable for the efficient delivery of such polypeptide compoundsto a wide variety of animal species because of the flexibility madepossible by the various substitutions at numerous positions of thepolypeptide compounds, by selecting the polar, neutral or non-polarnature of the C-terminal and center portions of these polypeptidecompounds so as to be compatible with the desired method of delivery isoral, nasal, continuous delivery utilizing special chemical/mechanicalmethods of delivery.

These peptides can be used therapeutically for any use for which growthhormone can be used such as treating hypothalamic pituitary dwarfism,osteoporosis, burns, and renal failure for acute use, for non-union bonefracture, and to promote wound healing. Additionally, it can be used topromote recovery from surgery, and acute/chronic debilitating medicalillnesses. Beneficial anabolic effects result on skin, muscle and bonein relation to the aging process with a concomitant decrease in bodyfat. Treatment of cancer patients by these peptides is also included,for example, prevention and/or reduction of cachexia in cancer patients.These therapeutic uses are accomplished by using a therapeuticallyeffective amount of the peptide. Such an amount is that needed topromote the release of serum growth hormone levels as discussed, infra.

The compounds of this invention may also be used to enhance blood GHlevels in animals; enhance milk production in cows; enhance body growthin animals such as mammals (e.g., humans, sheep, bovines, and swine), aswell as fish, fowl, other vertebrates and crustaceans; and increase wooland/or fur production in mammals. The amount of body growth is dependentupon the sex and age of the animal species, quantity and identity of thegrowth hormone releasing compound being administered, route ofadministration, and the like. Also, the compounds of this inventionincrease serum GH in humans; enhance body growth in short staturechildren; decrease body fat and improve protein metabolism in selectchildren; improve protein metabolism of the skin, muscle, bone whiledecreasing body fat of the elderly, particularly when GH deficiency ispresent.

These peptides are also useful for improving serum lipid pattern inhumans by decreasing in the serum the amount of serum cholesterol andlow density lipoprotein and increasing in the serum the amount of thehigh density lipoprotein.

The novel polypeptide compounds of this invention can be synthesizedaccording to the usual methods of solution and solid phase peptidechemistry, or by classical methods known in the art.

For the peptide amides, the solid-phase synthesis is preferred commencedfrom the C-terminal end of the peptide. A suitable starting material canbe prepared, for instance, by attaching the required protectedalpha-amino acid to a chloromethylated resin, a hydroxymethyl resin, abenzhydrylamine (BHA) resin, or a para-methyl-benzylhydrylamine(p-Me-BHA) resin. One such chloromethyl resin is sold under thetradename BIOBEADS SX-1 by Bio Rad Laboratories, Richmond, Calif. Thepreparation of the hydroxymethyl resin is described by Bodansky et al.,Chem. Ind. (London) 38, 1597 (1966). The BHA resin has been described byPietta and Marshall, Chem. Comm., 650 (1970) and is commerciallyavailable from Peninsula Laboratories, Inc., Belmont, Calif.

After the initial attachment, the alpha-amino protecting group can beremoved by a choice of acidic reagents, including trifluoroacetic acid(TFA) or hydrochloric acid (HCl) solutions in organic solvents at roomtemperature. After removal of the alpha-amino protecting group, theremaining protected amino acids can be coupled stepwise in the desiredorder. Each protected amino acid can be generally reacted in about a3-fold excess using an appropriate carboxyl group activator such asdicyclohexylcarbodiimide (DCC) or diisopropyl carbodiimide (DIG) insolution, for example, in methylene chloride (CH₂ Cl₂) ordimethylformamide (DMF) and mixtures thereof.

After the desired amino acid sequence has been completed, the desiredpeptide can be cleaved from the benzhydrylamine resin support bytreatment with a reagent such as hydrogen fluoride (HF) which not onlycleaves the peptide from the resin, but also cleaves most commonly usedside-chain protecting groups. When a chloromethyl resin or hydroxymethylresin is used, HF treatment results in the formation of the free peptideacid. However, peptide alkylamides and esters can readily be preparedfrom these peptide resins by cleavage with a suitable alkylamine,dialkylamine, or diaminoalkane or transesterification with an alcohol athigh pH levels.

The solid-phase procedure discussed above is well known in the art andhas been described by Stewart and Young, Solid Phase Peptide Synthesis:Second Edn. (Pierce Chemical Co., Rockford, Ill. 1984).

Some of the well known solution methods which dan be employed tosynthesize the peptide moieties of the instant invention are set forthin Bodansky et al., Peptide Synthesis, 2nd Edition, John Wiley & Sons,New York, N.Y. 1976.

It is believed that the peptides will be more preferably synthesized bya solution phase method which involves the condensation reaction of atleast two peptide fragments.

This method comprises condensing a peptide fragment X-A₁ -Y with thepeptide fragment U-V-W, wherein all amino acid side-chains except for A₁are neutral or protected, and wherein X is Prot. where Prot. isN-terminus protecting group; Y is A₂ -Q, Ala-A₂ -Q, A₂ -Ala-Q, an Ala-A₂-Ala-Q, A₂ -Ala-Trp-Q, Ala-A₂ -Ala-Trp-Q, Ala-Q or -Q, where when Y isQ, U is J-A₂ -Ala-Trp, or J-Ala-A₂ -Ala-Trp. When Y is Ala-Q, U is J-A₂-Ala-Trp. When Y is A₂ -Q or Ala-A₂ -Q, U is J-Ala-Trp. When Y is A₂-Ala-_(Q) or Ala-A₂ -A₃ -Q, U is J-Trp. When Y is A₂ -Ala-Trp-Q orAla-A₂ -Ala-Trp-Q, U is J. V is A₅ or Z. When V is A₅, W is Z. When V isZ, W is not present. A₁, A₂, A₅ and Z are as defined herein.

Q is the carboxy terminus of a peptide fragment and is -OR³ or -M, whereM is a moiety capable of being displaced by a nitrogen-containingnucleophile and R³ is H, an alkyl group containing one to about 10carbon atoms, an aryl group having from 6 to about 12 carbon atoms or anarylalkyl group having from 7 to about 12 carbon atoms; J represents theamine terminus of the indicated fragment and is H or a protecting group,which does not hinder the coupling reaction, for example, benzyl.

Thereafter, one removes the protecting groups. Alternatively, one mayuse the protected peptide thus formed in further condensations toprepare a larger peptide.

This preferred method is more fully described in U.S. patent applicationSer. No, 558,121 filed on Jul. 24, 1990, by John C. Hubbs and S. W.Parker entitled "Process for Synthesizing Peptides", published asWO92/01709 which is incorporated herein by reference.

In accordance with another embodiment of the present invention, a methodis provided for promoting release and/or elevation of growth hormonelevels in the blood of an animal. This method of promoting the releaseand/or elevation of growth hormone levels can also be used totherapeutically treat the aforesaid diseases. Said methods compriseadministering to an animal an effective dose of at least one of theabove-described polypeptides. In one embodiment this method is used inanimals other than humans.

The compounds of this invention can be administered by oral, parenteral(intramuscular (i.m.), intraperitoneal (i.p.), intravenous (i.v.) orsubcutaneous (s.c.) injection), nasal, vaginal, rectal or sublingualroutes of administration as well as intrapulmonary inhalation and can beformulated in dose forms appropriate for each route of administration.Parenteral administration is preferred.

Solid dose forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dose forms, the activecompound is mixed with at least one inert carrier such as sucrose,lactose, or starch. Such dose forms can also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets and pills, the dose forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dose forms for oral administration include emulsions, solutions,suspensions, syrups, the elixirs containing inert diluents commonly usedin the art, such as water. Besides, such inert diluents, compositionscan also include adjuvants, such as wetting agents, emulsifying andsuspending agents, and sweetening, flavoring, and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdose forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in a medium of sterile water, or some other sterileinjectable medium immediately before use.

The novel compounds of the present invention are also useful whenadministered in combination with growth hormone releasing hormone (i.e.,naturally occurring growth hormone releasing hormone, analogs andfunctional equivalents thereof), as well as in combination with othercompounds which promote the release of growth hormone, e.g., growthhormone releasing peptides (see U.S. Pat. No. 4,880,778 which isincorporated herein by reference). Such Combinations represent anespecially preferred means to administer the growth hormone releasingpeptides of the present invention because the combination promotes therelease of much more growth hormone than is predicted by the summationof the individual responses for each component of the combination, i.e.,the combination provides a synergistic response relative to theindividual component. Further details on the administration ofcombinations of growth hormone releasing peptides are described in theabove-cited patent. Such synergistic compounds are preferably compoundswhich act as an agonist at the growth hormone releasing hormone receptoror inhibit the effect of somatostatin. The synergism can be binary, i.e.the present compound and one of the synergistic compounds, or involvemore than one synergistic compound.

Combinations effective to cause the release and elevation of the levelof growth hormone in the blood of an animal such as humans comprise aneffective amount of polypeptides selected from the presently claimedpolypeptides and at least one of the following groups: Group 1polypeptides, or Group 2 polypeptides, wherein Group 1 polypeptides areselected from any of the naturally occurring growth hormone releasinghormones and functional equivalents thereof, wherein said polypeptidesact at the growth hormone releasing hormone receptor of mammals andother vertebrates, and crustaceans;

Group 2 polypeptides are selected from any of the polypeptides havingthe structure:

Tyr-DArg-Phe-NH₂ ;

Tyr-DAla-Phe-NH₂ ;

Tyr-DArg (NO₂)-Phe-NH₂ ;

Tyr-DMet (O)-Phe-NH₂ ;

Tyr-DAla-Phe-Gly-NH₂

Tyr-DArg-Phe-Gly-NH₂ ;

Tyr-DThr-Phe-Gly-NH₂ ;

Phe-DArg-Phe-Gly-NH₂ ;

Tyr-DArg-Phe-Sar-NH₂ ;

Tyr-DAla-Gly-Phe-NH₂ ;

Tyr-DArg-Gly-Trp-NH₂ ;

Tyr-DArg(NO₂)-Phe-Gly-NH₂ ;

Tyr-DMet (O)-Phe-Gly-NH₂

(NMe)Tyr-DArg-Phe-Sar-NH₂ ;

Tyr-DArg-Phe-Gly-ol;

Tyr-DArg-Gly-(NMe)Phe-NH₂ ;

Tyr-DArg-Phe-Sar-ol

Tyr-DAla-Phe-Sar-ol

Tyr-DAla-Phe-Gly-Tyr-NH₂ ;

Tyr-DAla-(NMe)Phe-Gly-Met(O)-ol;

Tyr-DArg-(NMe)Phe-Gly-Met(O)-ol;

Gly-Tyr-DArg-Phe-Gly-NH₂ ;

Tyr-DThr-Gly-Phe-Thz-NH₂ ;

Gly-Tyr-DAla-Phe-Gly-NH₂ ;

Tyr-DAla-Phe-Gly-ol;

Tyr-DAla-Gly-(NMe)Phe-Gly-ol;

Tyr-DArg-Phe-Sar-NH₂ ;

Tyr-DAla-Phe-Sar-NH₂ ;

Tyr-DAla-Gly-(NMe)Phe-NH₂ ;

Sar-Tyr-DArg-Phe-Sar-NH₂ ;

Tyr-DCys-Phe-Gly-DCys-NH₂ (cylic disulfide);

Tyr-DCys-Phe-Gly-DCys-NH₂ (free dithiol);

Tyr-DCys-Gly-Phe-DCys-NH₂ (cyclic disulfide);

Tyr-DCys-Gly-Phe-DCys-NH₂ (free dithiol);

Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH₂ ;

Tyr-DAla-Phe-Sar-Tyr-Pro-Ser-NH₂ ;

Tyr-DAla-Phe-Sar-Phe-Pro-Ser-NH₂ ;

Tyr-DAla-Phe-Gly-Tyr-Hyp-Ser-NH₂ ;

Tyr-DAla-Phe-Sar-Tyr-Hyp-Ser-NH₂ ;

Tyr-DAla-Phe-Sar-Phe-Hyp-Ser-NH₂ ;

Tyr-DArg-Phe-Gly-Tyr-Hyp-Ser-NH₂ ;

Tyr-DArg-Phe-Sar-Tyr-Pro-Ser-NH₂ ;

Tyr-DArg-Phe-Sar-Tyr-Hyp-Ser-NH₂ ;

Tyr-DArg-Phe-Gly-Tyr-Pro-Ser-NH₂ ; and

organic or inorganic addition salts of said polypeptides of Group 2;wherein said combination is administered in a ratio such that saidcombination is effective to cause the synergistic release and elevationof growth hormone in the blood of such animal.

In one preferred embodiment one uses Group 1 and Group 2 compounds alongwith the present peptides.

The amount of polypeptide or combination of polypeptides of the presentinvention administered will vary depending on numerous factors, e.g.,the particular animal treated, its age and sex, the desired therapeuticaffect, the route of administration and which polypeptide or combinationof polypeptides are employed. In all instances, however, a doseeffective (therapeutically effective amount) to promote release andelevation of growth hormone level in the blood of the recipient animalis used. Ordinarily, this dose level falls in the range of between about0.1 μg to 10 mg of total polypeptide per kg of body weight. Thepreferred amount can readily be determined empirically by the skilledartisan based upon the present disclosure.

For example, in humans when the mode of administration is i.v. thepreferred dose level falls in the range of about 0.1 μg to 10 μg oftotal polypeptide per kg of body weight, more preferably, about 0.5 μgto 5 μg of total polypeptide per kg of body weight, still morepreferably about 0.7 μg about 3.0 μg per kg of body weight. Whencombinations of growth hormone releasing peptides are used, loweramounts of the presently described peptide can be used. For example,combining the presently described peptide with, for example, asynergistic compound in Group I of U.S. Pat. No. 4,880,778 such as GHRH,a preferred range is about 0.1 μg to about 5 μg of the presentlydescribed compound per kg of body weight and about 0.5 μg to about 15.0μg of synergistic compound (e.g. GHRH) and more preferably about 0.1 μgto about 3 μg of the present compound with about 1.0 μg to about 3.0 μgof the synergistic compound per kg of body weight.

When the mode of administration is oral, greater amounts are typicallyneeded. For example, in humans for oral administration, the dose levelis typically about 30 μg to about 1200 μg of polypeptide per kg of bodyweight, more preferably about 70 μg to about 600 μg of polypeptide perkg of body weight, still more preferably, about 200 μg to about 600 μgof total polypeptide per kg of body weight. Cows and pigs require aboutthe same dose level as humans, while rats typically require higher doselevels. The exact level can readily be determined empirically based uponthe present disclosure.

In general, as aforesaid, the administration of combinations of growthhormone releasing peptides will allow for lower doses of the individualgrowth hormone releasing compounds to be employed relative to the doselevels required for individual growth hormone releasing compounds inorder to obtain a similar response, due to the synergistic effect of thecombination.

Also included within the scope of the present invention are compositionscomprising, as an active ingredient, the organic and inorganic additionsalts of the above-described polypeptides and combinations thereof;optionally, in association with a carrier, diluent, slow release matrix,or coating.

The organic or inorganic addition salts of the growth hormone releasingcompounds and combinations thereof contemplated to be within the scopeof the present invention include salts of such organic moieties asacetate, trifluoroacetate, oxalate, valerate, oleate, laurate, benzoate,lactate, rosylate, citrate, maleate, fumarate, succinate, tartrate,naphthalate, and the like; and such inorganic moieties as Group I (i.e.,alkali metal salts), Group II (i.e. alkaline earth metal salts) ammoniumand protamine salts, zinc, iron, and the like with counterions such aschloride, bromide, sulfate, phosphate and the like, as well as theorganic moieties referred to above.

Pharmaceutically acceptable salts are preferred when administration tohuman subjects is contemplated. Such salts include the non-toxic alkalimetal, alkaline earth metal and ammonium salts commonly used in thepharmaceutical industry including sodium, potassium, lithium, calcium,magnesium, barium, ammonium and protamine salts which are prepared bymethods well known in the art. The term also includes non-toxic acidaddition salts which are generally prepared by reacting the compounds ofthis invention with a suitable organic or inorganic acid. Representativesalts include hydrochloride, hydrobromide, sulfate, bisulfate, acetate,oxalate, valerate, oleate, laurate, borate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, and the like.

The invention will be further illustrated by the following non-limitingexamples.

EXAMPLE 1 Synthesis of The Growth Hormone Releasing Peptides

Paramethyl benzhydrylamine hydrochloride (pMe-BHA.HCl) resin is placedin a reaction vessel on a commercially available automated peptidesynthesizer. The resin is substituted with free amine up to a loading ofabout 5 mmoles per gram. The compounds are prepared by couplingindividual amino acids starting at the carboxy terminus of the peptidesequence using an appropriate activating agent, such asN,N'-dicyclohexylcarbodiimide (DCC). The alpha amine of individual aminoacids are protected, for example, as the t-butyloxycarbonyl derivative(t-Boc) and the reactive side chain functionalities are protected asoutlined in Table 1.

                  TABLE 1                                                         ______________________________________                                        Side Chain Protecting Groups Suitable For                                     Solid Phase Peptide Synthesis                                                 Arginine:        N.sup.g -Tosyl                                               Aspartic Acid:   O-Benzyl                                                     Cysteine:        S-para-Methylbenzyl                                          Glutamic Acid:   O-Benzyl                                                     Histidine:       N.sup.im -Tosyl                                              Lysine:          N.sup.ε -2,4-Dichlorobenzyloxycarbonyl               Methionine:      S-Sulfoxide                                                  Serine:          O-Benzyl                                                     Threonine:       O-Benzyl                                                     Tryptophan:      N.sup.in -Formyl                                             Tyrosine:        0-2,6-Dichlorobenzyl                                         ______________________________________                                    

Prior to incorporation of the initial amino acid, the resin is agitatedthree times (about one minute each) with dichloromethane (CH₂ Cl₂ ;about 10 mL/gm of resin), neutralized with three agitations (about twominutes each) of N,N-diisopropylethylamine (DIEA) in dichloromethane(10:90; about 10 mL/gm of resin) and agitated three times (about oneminute each) with dichloromethane (about 10 mL/gm of resin). The initialand each of the subsequent amino acids are coupled to the resin using apreformed symmetrical anhydride using about 6.0 times the total amountof the reaction capacity of the resin of a suitably protected amino acidand about 2.0 times the total amount of the binding capacity of theresin of DIG in an appropriate amount of dichloromethane. For aminoacids with a low dichloromethane solubility, N,N-dimethylformamide (DMF)is added to achieve a homogenous solution. Generally, the symmetricalanhydride is prepared up to 30 minutes prior to introduction into thereaction vessel at room temperature or below. The dicyclohexylurea thatforms upon preparation of the symmetrical anhydride is removed viagravity filtration of the solution into the reaction vessel. Progress ofthe coupling of the amino acid to the resin is commonly monitored via acolor test using a reagent such as ninhydrin (which reacts with primaryand secondary amines). Upon complete coupling of the protected aminoacid to the resin (>99%), the alpha amine protecting group is removed bytreatment with acidic reagent(s). A commonly used reagent consists of asolution of trifluoroacetic acid (TFA) in dichloromethane (33:66).

After the desired amino acid sequence has been completed, the desiredpeptide can be cleaved from the resin support by treatment with areagent such as hydrogen fluoride (HF) which not only cleaves thepeptide from the resin, but also cleaves most commonly used side-chainprotecting groups. When the BHA or p-Me-BHA resin is used, HF treatmentresults directly in free peptide amides. When a amino acid-Merrifieldresin is used, free peptide alkylamides are cleaved by treatment with anappropriate amine (in this case, use of Boc-N.sup.ε -FMOC-Lys wouldallow simultaneous removal of the FMOC group). The complete procedurefor incorporation of each individual amino acid residue onto the resinis outlined in Table 2.

                  TABLE 2                                                         ______________________________________                                        Procedure for Incorporation Of Individual                                     Amino Acids Onto a Resin                                                      Reagent                Agitations                                                                              Time/Agitation                               ______________________________________                                        1.    Dichloromethane  3         1     min.                                   2.    TFA-Dichloromethane                                                                            1         2     min.                                         (33:66)                                                                 3.    TFA-Dichloromethane                                                                            1         20    min.                                         (33:66)                                                                 4.    Dichloromethane  3         1     min.                                   5.    DIEA, DMF        2         2     min                                          (10:90)                                                                 6.    Dichloromethane  3         1     min.                                   7.    Boc amino acid/DIC                                                                             1         15-120                                                                              min.*                                  8.    Dichloromethane  3         1     min.                                   10.   Monitor progress of                                                           the coupling reaction**                                                 11.   Repeat steps 1-12 for each                                                    individual amino acid                                                   ______________________________________                                         *Coupling time depends upon the individual amino acid.                        **The extent of coupling can be generally monitored by a color test. If       the coupling is incomplete, the same amino acid can be recoupled by a         different protocol, eg HOBt active ester. If the coupling is complete the     next amino acid can then be coupled.                                     

Using this procedure the compounds described in Tables 3 and 4 weremade.

EXAMPLE 2 In Vivo GH Release In Rats

Immature female Sprague-Dawley rats were obtained from the Charles RiverLaboratories (Wilmington, Ma.). After arrival they were housed at 25° C.with a 14:10 hour light:dark cycle. Water and Purina rat chow wereavailable ad libitum. Pups were kept with their mothers until 21 days ofage.

Twenty-six day old rats, six rats per treatment group, were anesthetizedinterperitoneally with 50 mg/kg of pentobarbital 20 minutes prior toi.v. treatment with peptide. Normal saline with 0.1% gelatin was thevehicle for intravenous (i.v.) injections of the peptides. Theanesthetized rats, weighing 55-65 grams, were injected i.v. with thequantity of growth hormone releasing compounds indicated in Table 3.Injection was made as a 0.1 mL solution into the jugular vein.

All animals were sacrificed by guillotine 10 minutes after the finaltest injection (see Table 3). Trunk blood for the determination of bloodGH levels was collected following decapitation. After allowing the bloodto clot, it was centrifuged and the serum was separated from the clot.Serum was kept frozen until the day of sampling for radioimmunoassay(RIA) determination of growth hormone levels according to the followingprocedure, as developed by the National Institute of Arthritis, Diabetesand Digestive and Kidney Diseases (NIADDK).

Reagents are generally added to the RIA analysis tubes at a singlesitting, at refrigerator temperature (about 4° C.) in the followingsequence:

(a) buffer,

(b) "cold" (i.e., non-radioactive) standard or unknown serum sample tobe analyzed,

(c) radio-iodinated growth hormone antigen, and

(d) growth hormone antiserum.

Reagent addition is generally carried out so that there is achieved afinal RIA tube dilution of about 1:30,000 (antiserum to total liquidvolume; vol:vol).

The mixed reagents are then typically incubated at room temperature(about 25° C.) for about 24 hours prior to addition of a second antibody(e.g., goat or rabbit anti-monkey gamma globulin serum) which binds toand causes precipitation of the complexed growth hormone antiserum.Precipitated contents of the RIA tubes are then analyzed for the numberof counts in a specified period of time in a gamma scintillationcounter. A standard curve is prepared by plotting number of radioactivecounts versus growth hormone (GH) level. GH levels of unknowns are thendetermined by reference to the standard curve.

Serum GH was measured by RIA with reagents provided by the NationalHormone and Pituitary Program.

Serum levels in Table 3 are recorded in ng/mL in terms of the rat GHstandard of 0.61 International Units/mg (IU/mg). Data is recorded as themean+/- standard error of the mean (SEM). Statistical analysis wasperformed with Student's t-test. In Table 3 the results shown are theaverage of studies with six rats.

                                      TABLE 3                                     __________________________________________________________________________    In Vivo GH Release Prompted By Growth                                         Hormone Releasing Compounds In Pentobarbital Anesthetized Rats                (Animals Sacrificed 10 Minutes After Final Injection)                         Column A          Total                                                                             Control Serum                                                                         GH Released                                     GH Releasing      Dose.sup.a                                                                        GH ng/ml +                                                                            Serum GH ng/ml +                                Peptide           (μg/iv)                                                                        SEM (N = 6)                                                                           SEM (N = 6)                                     __________________________________________________________________________    Ala--His-DβNal--                                                                           .1  337 ± 51                                                                           610 ± 90                                     Ala--Trp-DPhe--Lys--NH.sub.2.sup.b                                                              .3  337 ± 51                                                                           1140 ± 187                                                     1.0 337 ± 51                                                                           2909 ± 257                                                     3.0 337 ± 51                                                                           3686 ± 436                                   His-DTrp--Ala--Trp-                                                                             .1  131 ± 43                                                                            540 ± 148                                   DPhe--Lys--NH.sub.2.sup.b                                                                       .3  131 ± 43                                                                           751 ± 88                                                       1.0 131 ± 43                                                                           1790 ± 252                                                     3.0 131 ± 43                                                                           2481 ± 209                                   DAla-DβNal--Ala--                                                                          .1  337 ± 51                                                                           1381 ± 222                                   Trp-DPhe--Lys--NH.sub.2                                                                         .3  337 ± 51                                                                           2831 ± 304                                                     1.0 337 ± 51                                                                           2886 ± 179                                                     3.0 337 ± 51                                                                           3678 ± 287                                   Ala-DβNal--Ala--                                                                           0.3 167 ± 46                                                                           363 ± 73                                     Trp-DPhe--Lys--NH.sub.2                                                                         1.0 167 ± 46                                                                           1450 ± 294                                                     3.0 167 ± 46                                                                           2072 ± 208                                                     10.0                                                                              167 ± 46                                                                           2698 ± 369                                   DAla-LβNal--Ala--                                                                          .1          263 ± 45                                     Trp-DPhe--Lys--NH.sub.2                                                                         .3   160 ± 151                                                                         315 ± 85                                                       1.0  160 ± 151                                                                         426 ± 41                                     DAla-DTrp--Ala--Trp-                                                                            .1  111 ± 24                                                                           526 ± 86                                     DPhe--Lys--NH.sub.2                                                                             .3  111 ± 24                                                                           1608 ± 204                                                     1.0 111 ± 24                                                                           2820 ± 346                                                     3.0 111 ± 24                                                                           2437 ± 214                                   Ala-DβNal--Ala--                                                                           .1  167 ± 46                                                                           144 ± 20                                     NMeTrp-DPhe--Lys--NH.sub.2                                                                      0.3 167 ± 46                                                                           258 ± 28                                                       1.0 167 ± 46                                                                           261 ± 24                                                       3.0 167 ± 46                                                                           277 ± 85                                     D-Leu-DβNal--                                                                              .1  160 ± 51                                                                           256 ± 94                                     Ala--Trp-DPhe--Lys--NH.sub.2                                                                    .3  160 ± 51                                                                           452 ± 49                                                       1.0 160 ± 51                                                                           355 ± 94                                     D-Trp-DβNal--Ala--                                                                         .1  160 ± 51                                                                           226 ± 61                                     Trp-DPhe--Lys--NH 2                                                                             .3  160 ± 51                                                                           245 ± 27                                                       1.0 160 ± 51                                                                           437 ± 62                                     Ala--His-DβNal--Ala-                                                                       .3  160 ± 51                                                                           1418 ± 302                                   Trp-DPhe--Lys--NH.sub.2                                                                         1.0 160 ± 51                                                                           2201 ± 269                                   His-DTrp-Ala--Trp .1  140 ± 10                                                                           200 ± 40                                     DPhe--Lys--NH.sub.2.sup.b                                                                       .3  140 ± 10                                                                           505 ± 50                                                       1.0 140 ± 10                                                                           1640 ± 215                                   DAsn-DβNal--Ala--                                                                          .1  228 ± 23                                                                           122 ± 38                                     Trp-DPhe--Lys--NH.sub.2                                                                         .3  228 ± 23                                                                           195 ± 21                                                       1.0 228 ± 23                                                                           197 ± 47                                     DHis-DβNal--Ala--                                                                          .1  228 ± 23                                                                           386 ± 81                                     Trp-DPhe--Lys--NH.sub.2                                                                         .3  228 ± 23                                                                           605 ± 82                                                       1.0 228 ± 23                                                                           930 ± 96                                     DLys-DβNal--Ala--                                                                          .1  228 ± 23                                                                           262 ± 31                                     Trp-DPhe--Lys--NH.sub.2                                                                         .3  228 ± 23                                                                           340 ± 86                                                       1.0 228 ± 23                                                                           335 ± 56                                     DSer-DβNal--Ala--                                                                          .1  228 ± 23                                                                           226 ± 11                                     Trp-DPhe--Lys--NH.sub.2                                                                         .3  228 ± 23                                                                           171 ± 48                                                       1.0 228 ± 23                                                                           212 ± 43                                     Ala--His-DβNal--Ala--                                                                      .3  228 ± 23                                                                           1746 ± 318                                   Trp-DPhe--Lys--NH.sub.2                                                                         1.0 228 ± 23                                                                           2610 ± 176                                   Gly-DβNal--Ala--Trp-                                                                       .3  160 ± 36                                                                           1237 ± 249                                   DPhe--Lys--NH.sub.2                                                                             1.0 160 ± 36                                                                           2325 ± 46                                                      3.0 160 ± 36                                                                           2694 ± 370                                                     10.0                                                                              160 ± 36                                                                           3454 ± 159                                   Ser-DβNal--Ala--                                                                           .3  160 ± 36                                                                           227 ± 39                                     Trp-DPhe--Lys--NH.sub.2                                                                         1.0 160 ± 36                                                                            595 ± 112                                                     3.0 160 ± 36                                                                           1303 ± 281                                                     10.0                                                                              160 ± 36                                                                           2919 ± 320                                   Met-DβNal--Ala--                                                                           .3  160 ± 36                                                                           181 ± 48                                     Trp-DPhe--Lys--NH.sub.2                                                                         1.0 160 ± 36                                                                           226 ± 58                                                       3.0 160 ± 36                                                                           316 ± 66                                                       10.0                                                                              160 ± 36                                                                           1010 ± 236                                   Ala--His-DβNal--Ala--                                                                      0.1 160 ± 36                                                                            822 ± 243                                   Trp-DPhe--Lys--NH.sub.2.sup.b                                                                   0.3 160 ± 36                                                                           1594 ± 292                                                     1.0 160 ± 36                                                                           2180 ± 284                                   Gln-DβNal--Ala--Trp-                                                                       0.3 131 ± 43                                                                           124 ± 15                                     DPhe--Lys--NH.sub.2                                                                             1.0 131 ± 43                                                                           340 ± 66                                                       3.0 131 ± 43                                                                            476 ± 109                                                     10.0                                                                              131 ± 43                                                                            673 ± 228                                   Pro-DβNal--Ala--Trp-                                                                       0.3 135 ± 32                                                                           264 ± 31                                     DPhe--Lys--NH.sub.2                                                                             1.0 135 ± 32                                                                            513 ± 123                                                     3.0 135 ± 32                                                                           1690 ± 103                                   Gly-DβNal--Ala--Trp-                                                                       0.3 215 ± 33                                                                           1301 ± 260                                   DPhe--Lys--NH.sub.2                                                                             1.0 215 ± 33                                                                           2211 ± 146                                                     3.0 215 ± 33                                                                           2364 ± 365                                   NαAcetyl-Gly-                                                                             0.3  262 ± 53*                                                                          268 ± 21*                                   DβNal--Ala--Trp-DPhe--                                                                     1.0  262 ± 53*                                                                          599 ± 219*                                  Lys--NH.sub.2     3.0  262 ± 53*                                                                          626 ± 210*                                  Sar-DβNal--Ala--Trp-                                                                       0.3  262 ± 53*                                                                          908 ± 264*                                  DPhe--Lys--NH.sub.2                                                                             1.0  262 ± 53*                                                                          1681 ± 262*                                                   3.0  262 ± 53*                                                                          2600 ± 316*                                 DβNal--Ala--Trp-DPhe--                                                                     0.3 215 ± 33                                                                           436 ± 98                                     Lys--NH.sub.2     1.0 215 ± 33                                                                            660 ± 151                                                     3.0 215 ± 33                                                                            776 ± 274                                   NαAcetyl-DβNal--Ala--                                                                0.3  262 ± 53*                                                                          339 ± 17*                                   Trp-DPhe--Lys--NH.sub.2                                                                         1.0  262 ± 53*                                                                          430 ± 136*                                                    3.0  262 ± 53*                                                                          634 ± 118*                                  NαIsopropyl-DβNal--Ala--                                                             0.3  262 ± 53*                                                                          541 ± 179*                                  Trp-DPhe--Lys--NH.sub.2                                                                         1.0  262 ± 53*                                                                          972 ± 247*                                                    3.0  262 ± 53*                                                                          1636 ± 371*                                 Nαdiethyl-DβNal--Ala--                                                               0.3  127 ± 32*                                                                          462 ± 132*                                  Trp-DPhe--Lys--NH.sub.2                                                                         1.0  127 ± 32*                                                                          899 ± 160*                                                    3.0  127 ± 32*                                                                          1786 ± 373*                                 Nαethyl-DβNal--Ala--                                                                 0.3 135 ± 32                                                                           531 ± 80                                     Trp-DPhe--Lys--NH.sub.2                                                                         1.0 135 ± 32                                                                           1156 ± 250                                                     3.0 135 ± 32                                                                           2664 ± 225                                   Gly-DβNal--Ala--Trp-                                                                       0.3 135 ± 32                                                                           1387 ± 352                                   DPhe--Lys--NH.sub.2                                                                             1.0 135 ± 32                                                                           1958 ± 353                                                     3.0 135 ± 32                                                                           2605 ± 97                                    βAla-DβNal--Ala--Trp-                                                                 0.3 135 ± 32                                                                           1937 ± 343                                   DPhe--Lys--NH.sub.2                                                                             1.0 135 ± 32                                                                           3603 ± 645                                                     3.0 135 ± 32                                                                           4000 ± 500                                   Ava**-DβNal--Ala--                                                                         0.3 135 ± 32                                                                           2469 ± 185                                   Trp-DPhe--Lys--NH.sub.2                                                                         1.0 135 ± 32                                                                           4034 ± 680                                                     3.0 135 ± 32                                                                           3142 ± 392                                   Ala-DβNal--Ala--Trp-                                                                       0.3  208 ± 148*                                                                         211 ± 27*                                   DPhe--NHCH.sub.2 CH.sub.2 NH.sub.2                                                              1.0  208 ± 148*                                                                         468 ± 127*                                                    3.0  208 ± 148*                                                                         877 ± 325*                                                    30.0                                                                               208 ± 148*                                                                         2325 ± 477*                                 Ala-DβNal--Ala--Trp-DPhe--                                               NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub. NH.sub.2                                          0.3  208 ± 148*                                                                         284 ± 132*                                                    1.0  208 ± 148*                                                                         527 ± 166*                                                    3.0  208 ± 148*                                                                         816 ± 289*                                                    30.0                                                                               208 ± 148*                                                                         3650 ± 772*                                 D-Ala-DβNal--Ala--Trp-DPhe--                                                               0.3 111 ± 24                                                                           180 ± 37                                     NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 NH.sub.2                                         1.0 111 ± 24                                                                            686 ± 135                                                     3.0 111 ± 24                                                                           1490 ± 179                                                     10.0                                                                              111 ± 24                                                                           2248 ± 70                                    Ala-DβNal--Ala--Trp-                                                                       0.3  208 ± 148*                                                                         211 ± 48*                                   DPhe--OMe         1.0  208 ± 148*                                                                         157 ± 35*                                                     3.0  208 ± 148*                                                                         492 ± 147*                                                    30.0                                                                               208 ± 148*                                                                         554 ± 127*                                  D-Ala-DTrp--Ala-- 0.1 111 ± 24                                                                           526 ± 86                                     Trp-DPhe--Lys--NH.sub.2                                                                         0.3 111 ± 24                                                                           1608 ± 204                                                     1.0 111 ± 24                                                                           2820 ± 346                                                     3.0 111 ± 24                                                                           2437 ± 214                                   Aib-DβNal--Ala--Trp-                                                                       0.1  208 ± 148*                                                                         269 ± 58*                                   DPhe--Lys--NH.sub.2                                                                             0.3  208 ± 148*                                                                         331 ± 108*                                                    1.0  208 ± 148*                                                                         368 ± 133*                                                    3.0  208 ± 148*                                                                         1090 ± 176*                                 D-Ala-DβNal--Ala--Trp--DPhe--                                                              0.1 215 ± 49                                                                            608 ± 115                                   Lys(ξ1Pr)--NH.sub.2                                                                          0.3 215 ± 49                                                                           1753 ± 419                                                     1.0 215 ± 49                                                                           1817 ± 297                                                     3.0 215 ± 49                                                                           2336 ± 196                                   __________________________________________________________________________     *Dissolved in DMSO,                                                           **Dissolved in aminovaleric acid (Ava)                                        .sup.a Doses so noted were administered to 29 day old female rats.            .sup.b Control GHRP's                                                    

In Table 3, compounds of the invention are compared to compounds outsideof the present generic formula and shown to promote the release andelevation of growth hormone levels in the blood of rats to which suchcompounds have been administered in a superior fashion. The surprisinggrowth hormone releasing activity of the preferred compounds are quitevaluable since a shorter-chain, lower molecular weight polypeptide withthe relatively stable and inexpensive amino acid D-alanine at theamino-terminus and pentanediamine in place of Lys at the C-terminusshould prove to be a low cost means to enhance growth hormone levels inanimals and humans.

EXAMPLE 3 - In Vivo GH Release In Rats After Oral Administration

The procedure of Example 2 was repeated, except the rats were givenindicated doses of compounds by intragastric tubes. The compoundsadministered, the dose levels employed and results are set forth inTable 4.

                                      TABLE 4                                     __________________________________________________________________________    In Vivo GH Release Promoted By Growth                                         Hormone Releasing Compounds In Pentobarbital Anesthetized Rats                (Rats sacrificied at various times after intragastric administration of       peptide)                                                                                                  Serum GH nb/ml ±                                                           SEM (N = 6 at:)                                   Column A    Total                                                                              Serum GH ng/ml ±                                                                      15'                                               GH Releasing                                                                              Dose SEM        (20')                                             Peptide     (mg/kg)                                                                            (N = 6)    (30')                                             __________________________________________________________________________    Ala--His-DβNal--Ala--                                                                10   247 ±                                                                           32    786 ±                                                                           244                                          Trp-DPhe--Lys--NH.sub.2.sup.a                                                             30   247 ±                                                                           32    1914 ±                                                                          294                                          DAla-DβNal--Ala--                                                                     10* 247 ±                                                                           32    116 ±                                                                           298                                          Trp-DPhe--Lys--NH.sub.2                                                                    30**                                                                              247 ±                                                                           32    2038 ±                                                                          444                                          Ava-DβNal--Ala--                                                                     30   322 ±                                                                           145*  2135 ±                                                                          586*                                         Trp-DPhe--Lys--NH.sub.2                                                       Gly-DβNal--Ala--                                                                     30   247 ±                                                                           32    1072 ±                                                                          137                                          Trp-DPhe--Lys--NH.sub.2                                                       His-DTrp--Ala--                                                                           30   247 ±                                                                           32    1810 ±                                                                          437                                          Trp-DPhe--Lys--NH.sub.2.sup.a                                                 Ala--His-DβNal--Ala--                                                                10   196 ±                                                                           49 (15')                                                                            1421 ±                                                                          363                                          Trp-DPhe--Lys--NH.sub.2.sup.a                                                                  147 ±                                                                           30 (20)                                                                             1605 ±                                                                          621 (20')                                                     133 ±                                                                           18 (30')                                                                            752 ±                                                                           81 (30')                                     DAla-DβNal--Ala--                                                                    10   196 ±                                                                           49 (15')                                                                            706 ±                                                                           133 (15')                                    Trp-DPhe--Lys--NH.sub.2                                                                        147 ±                                                                           30 (20')                                                                            1062 ±                                                                          254 (20')                                    Gly-DβNal--Ala--                                                                     10   196 ±                                                                           49 (15')                                                                            957 ±                                                                           188 (15')                                    Trp-DPhe--Lys--NH.sub.2                                                                        147 ±                                                                           30 (20')                                                                            1685 ±                                                                          524 (20')                                    His-DTrp--Ala--                                                                           10   196 ±                                                                           49 (15')                                                                            1131 ±                                                                          189 (15')                                    Trp-DPhe--Lys.sup.a                                                                            147 ±                                                                           30 (20')                                                                            686 ±                                                                           149 (20')                                    βAla--DβNal--Ala                                                                10   196 ±                                                                           49 (15')                                                                            1202 ±                                                                          429 (15')                                    Trp-DPhe--Lys--NH.sub.2                                                                        147 ±                                                                           30 (20')                                                                            1217 ±                                                                          239 (20')                                    Ava-DβNal--Ala--                                                                     10   196 ±                                                                           49 (15')                                                                            1407 ±                                                                          204 (15')                                    Trp-DPhe--Lys--NH.sub.2                                                                        147 ±                                                                           30 (20')                                                                            1251 ±                                                                          351 (20')                                    __________________________________________________________________________     *Suspension (light* or heavy**)acetic acid added.                             .sup.a Control peptides                                                       The compounds of the present invention retain useful levels of GH             releasing activity after oral administration to rats. This is valuable        since therapeutic usefulness of the peptides is enhanced by this method o     administration.                                                          

EXAMPLE 4 Condensation Reaction of Peptide Fragments To Form Peptide

General Procedures

Melting points can be determined using a Thomas Hoover capillary meltingpoint apparatus. Infrared (IR) spectra can be recorded on a Perkin-ElmerModel 137 or a Nicolet Model 5DX spectrophotometer and reported in wavenumbers (cm⁻¹). Mass spectra (MS) can be obtained using a VG AnalyticalLtd. Model ZAB-1F Mass Spectrometer in EI (electron impact), FD (fielddesorption) or FAB (fast atom bombardment) modes. GCMS can be obtainedusing a Finnigan 4023 GCMS equipped with a 30 m DB5 capillary column (J& W Scientific) using helium carrier gas. Optical rotations can bemeasured using an Autopol III polarimeter manufactured by RudolphResearch.

¹ H NMR spectra can be obtained on a JEOL GX-400 NMR instrumentoperating at 400 MHz or a JEOL GX-270 NMR instrument operating at 270MHz. These instruments are capable of a routine digital resolution ofless than 0.7 Hz. Chemical shifts are expressed in parts per millionrelative to internal 3-(trimethylsilyl)-tetradeutero sodium propionate(TSP).

High performance liquid chromatography (HPLC) can be accomplished usinga Hitachi system consisting of a L-5000 gradient controller and a 655Apump attached to a Vydac 201TP1010 or 218TP1010 semipreparative column.Combinations of water containing 0.2% trifluoroacetic acid and methanolcan be used as the eluting solvent. Typically, compounds of interestwill be eluted at a flow rate of six mL per minute with a gradientincreasing the organic component at a rate of approximately 1-2% perminute. Compounds are then detected at appropriate wavelengths using anLKB 2140 diode array U.V. detector. Integrations can then beaccomplished using Nelson Analytical software (Version 3.6).

Reactions will be carried out under an inert atmosphere of nitrogen orargon unless otherwise specified. Anhydrous tetrahydrofuran (THF, U.V.grade) and dimethylformamide (DMF) can be purchased from Burdick andJackson and used directly from the bottle.

A. Preparation of Tripeptide Fragment - ₂ HN-Trp-DPhe-Lys(Boc)-NH₂

N.sup.α -Benzyloxycarbonyl-(N.sup.ε -t-butoxycarbonyl)lysine amide, 4.

To a 10° C. solution of carbonyldiimidazole (CDI, 2, 88.24 g, 0.544 mol)and dry tetrahydrofuran (THF, 1500 mL), N.sup.α-benzyloxycarbonyl-(N.sup.ξ -t-butoxycarbonyl)lysine (1, 180 g, 0.474mol), are slowly added. Gas evolution is observed during this addition.While the N.sup.ξ -benzyloxycarbonyl-(N.sup.ξ -t-butoxycarbonyl)lysineimidazolide intermediate, 3, is forming, a saturated solution of ammoniaand THF (2000 mL) is prepared (anhyd. NH₃ gas is passed through THF at5°-10° C.). After formation of intermediate 3 is judged to be complete(when gas evolution has ceased, approximately 2 hours), one-half of theTHF solution containing 3 is added to the ammonia solution. Theremainder of the solution containing 3 is added 30 minutes later. Acontinuous flow of ammonia gas is maintained throughout the additionsand for an additional 45 minutes thereafter. Upon addition of the twosolutions containing 3, a white precipitate forms. The reaction isallowed to warm to room temperature and to stir for 15 hours. Solvent isremoved from the slurry in vacuo. The residue is slurried in water, andthe resulting solid is collected by vacuum filtration.

N.sup.ξ -t-Butoxycarbonyl-lysine-amide, 5.

A solution of the lysine amide 4 (181.48 g, 0.479 mol) in methanol(MeOH, 1000 mL) is added to a catalyst slurry of 5% Pd/C (5 g) inmethanol (250 mL) under argon. Hydrogen is bubbled through the reactionmixture (ca. 15 minutes) and the reaction is then stirred under anatmosphere of hydrogen until HPLC analysis indicates that the reactionis complete (36 hours). The hydrogen atmosphere is then displaced withargon. The reaction solution is clarified through a Celite® pad andsolvent is removed in vacuo to provide a solid.

N.sup.α -Benzyloxycarbonyl-D-phenylalanyl-(N.sup.ξ -t-butoxtcarbonyl)lysine-amide, 8.

N.sup.α -Benzyloxycarbonyl-D-phenylalanine (6, 126.39 g, 0.423 mol) isslowly added to a 10° C. solution of CDI (2, 66.03 g, 0.409 mol) in THF(500 mL). Gas evolution is observed during the addition. When gasevolution ceases, the lysine amide 5 (110.75 g, 0.452 mol) is added as asolution in THF (500 mL). After approximately 48 hours the mixture isfiltered to remove solids. The filtrate is concentrated in vacuo.

The resulting residue is taken up in ethyl acetate (EtOAc, 500 mL) andis then washed as follows in a separatory funnel:

1. aq HCl (1 N, 3×500 mL) pH of wash 1, ca. 8; subsequent wash pH's, 1

2. water (500 mL),

3. aq Na₂ CO₃ (1/2 saturated, 2×500 mL), is filtered to collect theformed crystalline solids (8),

4. Water (3×500 mL).

The organic layer is dried over MgSO₄. After clarification, the solventis removed in vacuo. The resulting residue can be recrystallized fromhot EtOAc to provide a second sample of 8.

D-Phenylalanyl-(N.sup.ξ -t-butoxycarbonyl)lysine-amide, 9.

A methanolic solution (1500 mL) of amide 8 (120.53 g, 0.229 mol) isadded to a catalyst slurry of 5% Pd/C (50 g) in MeOH (200 mL). The argonatmosphere is displaced with hydrogen. When HPLC analysis indicates thatthe reaction is complete (ca. 4 hours), the hydrogen atmosphere isdisplaced with argon. The reaction solution is then clarified through aCelite® pad and the filtrate is taken to a residue in vacuo. Thisdipeptide product can be used directly in the preparation of tripeptide12.

N.sup.α -Benzyloxycarbonyl-tryptophyl-D-phenylalanyl-(N.sup.ξ-t-butoxycarbonyl)lysine-amide, 12.

A 10° C. solution of N.sup.α -benzyloxycarbonyl-tryptophan (10, 67.60 g,0.200 mol), THF (500 mL), and CDI (2, 33.05 g, 0.204 mol) is stirreduntil gas evolution ceases. A solution of 9 (40.8 g, 0.103 mol) in THF(ca. 200 mL) is then added to the reaction mixture. The resultingsolution is allowed to react for 15 hours while warming to roomtemperature. The solid which forms is then collected by vacuumfiltration. The filtrate is taken to a residue by concentration invacuo. The resulting residue and solid are recombined and taken up inEtOAc (4000 mL) with slight warming. Upon cooling the solution to roomtemperature, a solid forms. The solid is collected by vacuum filtration.This solid is recrystallized from hot MeOH to afford purified tripeptide12. The EtOAc filtrate (from the first crystallization) is washed asfollows in a separatory funnel:

1. aq HCl (1 N, 2×500 mL),

2. water (1×500 mL),

3. aq Na₂ CO₃ (1/2 saturated, 2×500 mL),

4. aq NaCl (1×500 mL).

The organic layer is dried over MgSO₄ and then clarified by vacuumfiltration. The solvent of the filtrate is removed in vacuo. Theresulting residue is again taken up in EtOAc to afford a dry solid. Thesolid can be subjected to a hot MeOH recrystallization to afford asecond crop of 12 as a white solid.

Tryptophyl-D-phenylalanyl-(N.sup.ξ -t-butyloxycarbonyl)lysine-amide 13.

A methanolic solution (1500 mL) of tripeptide 12 (64.59 g, 0.091 mol) isadded to a catalyst slurry of 5% Pd/C (5 g) and MeOH (250 mL) under anargon atmosphere. An additional volume of MeOH (2250 mL) is added. Theargon atmosphere is displaced with hydrogen and allowed to react (ca. 24hours). Upon completion of the reaction, the hydrogen atmosphere isdisplaced with argon. The solution is clarified through a Celite® padand the filtrate is concentrated in vacuo to provide tripeptide 13 as awhite solid.

B. Preparation of Tripeptide Fragment-K-DAla-D.sup.β Nal-Ala-OMe

N.sup.α -benzyloxycarbonyl-D-Alanyl-D-beta-napthyl alanine methyl ester,25

A solution of EtOAc (400 mL) and D-beta-napthylalanine methyl esterhydrochloride (22, 0.62 mol) are washed with saturated sodium carbonate(400 mL) and 0.8 N aqueous sodium hydroxide (ca. 500 mL). The resultingaqueous phase is removed (pH 8.5) and the organic phase is sequentiallywashed with half-saturated aqueous Na₂ CO₃ (150 mL) and then with water(50 mL). The free base form of 22 is isolated upon concentration of theethyl acetate layer in vacuo.

Dicyclohexylcarbodiimide (DCC, ca. 95 g, 0.46 mol) is added to a -5° C.(ice-ethanol bath) solution of N.sup.α -benzyloxycarbonyl-D-alanine (19,143.5 g, 0.50 mol), N-hydroxysuccinimide (HONSu, 23, 0.62 mol) and thefreshly prepared free base form of 22 (ca. 0.52 mol) in DMF (ca. 3 L).The resulting reaction solution is allowed to stir for 24 hours whilewarming to room temperature. HPLC analysis should be used to see if thereaction is complete. If it is not, the reaction solution is then cooledto ca. -5° C. and an additional portion of dicyclohexylcarbodiimide (ca.0.17 mol) is added to the reaction. The reaction mixture is then allowedto stir for an additional 24 hours while warming to room temperature.The mixture is then filtered to remove dicyclohexylurea (DCU). Water (1L) is added to the filtrate and the resulting solution is concentratedin vacuo. The resulting residue is taken up in aqueous 1N HCl (ca. 1 Luntil the pH of the aqueous phase reaches a pH of 1). The aqueous phaseis then extracted with two portions of ethyl acetate (1 L each). Theethyl acetate layers are discarded. The pH of the aqueous phase is thenadjusted by addition of cold 2N sodium hydroxide (500 mL) and sodiumhydroxide pellets. During this neutralization, the solution is kept coldby addition of cold ethyl acetate (1 L). When the pH of the aqueousphase reaches approximately 7, copious precipitation of a white solid oroil usually results. This precipitate is collected by vacuum filtrationor decantation and washed sequentially with half saturated sodiumcarbonate (2×1500 mL), water (6×1500 mL) and ethyl acetate (3×1500 mL).The resulting material is dried under high vacuum to constant weight.This material can be hydrolyzed directly without further purification.

N.sup.α -Benzyloxycarbonyl-D-alanyl-β-napthyl-D-alanine, 26.

Aqueous sodium hydroxide (192 mL, 0.08 g/mL solution, 0.38 mol) is addedto a solution of dipeptide 25 (ca. 0.38 mol), water (360 mL) and MeOH(ca. 6 L). The solution is stirred at room temperature until hydrolysisis complete (ca. 24 hours). The disappearance of the starting peptide isestablished by HPLC analysis. The solution is concentrated in vacuo to aresidue which is dissolved in water (ca. 1 L). The aqueous layer (pH ca.10) is then extracted with EtOAc (2×500 mL) in a separatory funnel. Theethyl acetate layers are discarded. The resulting aqueous phase isadjusted to a pH of approximately 5 with concentrated HCl at which pointprecipitation of a white solid or oil usually results. The product iscollected and is dried in vacuo.

N.sup.α -Benzyloxycarbonyl-D-alanyl-D-beta-napthyl alanyl-alanine methylester, 20.

The dipeptide N.sup.α -benzyloxycarbonyl-D-alanyl-D-beta-napthyl alanine(26, 0.253 mol) is added to a solution of the HONSu (23, 0.505 mol) inDMF (800 mL) under an atmosphere of argon. To this solution, a mixtureof alanine methyl ester hydrochloride (15, 0.303 mol),N-methylmorpholine (16, 0.303 mol) and DMF (200 mL) is added. Theresulting solution is cooled to 10° C., at which timedicyclohexylcarbodiimide (24, 0.265 mol) in methylene chloride (273 mL)is added. The reaction is monitored by HPLC while the reactiontemperature is maintained at 10° C. until the reaction is complete. Ifafter several days (ca. 4), the reaction has not progressed tocompletion, an additional charge of 24 (0.080 mol) is added and thereaction mixture is allowed to stir for an additional day at 10° C. Thereaction is again monitored by HPLC analysis until complete (typicallyca. 5 days). The solids which form during the reaction are collected byvacuum filtration. The filtrate is then concentrated to a residue invacuo. The resulting residue is taken up in ethyl acetate and extractedwith half-saturated aqueous Na₂ CO₃ (2×500 mL). The ethyl acetate phaseis dried over MgSO₄. The resulting solution is clarified and isconcentrated to a residue in vacuo.

A 2N aqueous sodium hydroxide solution (7.5 mL, 15 mmol) is added to amethanol (500 mL) and water solution (200 mL) containing N.sup.α-Benzyloxycarbonyl-DAla-D.sup.β Nal-Ala-OMe (13.7 mmol). After thereaction is allowed to stir overnight at room temperature, HPLC analysisindicates the amount of the starting material remaining. When it isessentially complete (ca. overnight), the resulting solution isconcentrated in vacuo to a volume of approximately 200 mL. Water (100mL) is added and the pH is adjusted to approximately 12 by addition of2N sodium hydroxide (1 mL). The resulting solution is extracted withethyl acetate (2×500 mL). The ethyl acetate layers are discarded. The pHof the aqueous phase is then adjusted to approximately 5 by addition ofaqueous HCl which usually results in the precipitation of the product.It is important to minimize the volume of the aqueous phase to promotethis precipitation. The aqueous phase is decanted away from the productand the product is then rinsed with water (2×50 mL). The isolatedproduct is dried to constant weight in vacuo.

C. Condensation Reaction Of Peptide Fragments To Produce Hextapeptide

The two peptides DAla-D.sup.β Nal-Ala-OH (33, 2.6 mmol) andTrp-D-Phe-Lys(Boc)-NH₂ (13, 2.8 mmol) are dissolved in anhydrous DMF andthe resulting solution is concentrated in vacuo. This preliminaryconcentration is carried out in an attempt to remove any traces ofmethanol which might be present. The resultant peptide mixture isredissolved in DMF and N-hydroxysuccinimide (5.1 mmol) is then added.The resulting solution is then cooled to a solution temperature of -2°C. and dicyclohexylcarbodiimide (3.4 mmol) is then added as a solutionin methylene chloride (3.5 mL). The resulting reaction mixture isallowed to stir at -2° C. solution temperature for a period of threedays. HPLC analysis is used to determine if the reaction is essentiallycompleted. After this period of time, if it is not, additionaldicyclohexylcarbodiimide can then be added and the resultant reactionmixture allowed to stir for an additional day at -2° C. If, on thefollowing day (for a total of four days) HPLC analysis again indicatesincomplete reaction, cooling of the reaction mixture should beterminated. The solution temperature of the reaction can be allowed toslowly rise to room temperature (25° C.) over a period of hours (ca. 8).The resultant reaction mixture is allowed to stir overnight at roomtemperature. The procedure is repeated until the reaction is complete.Then, water (50 mL) is added and the resulting mixture is allowed tostir for an additional day. The reaction solution is then filtered toremove dicyclohexylurea and the resulting filtrate is concentrated invacuo to a viscous oil. Ethyl acetate and half-saturated aqueous sodiumcarbonate (200 mL) are added to the resulting residue. The two-phasemixture is vigorously swirled on a rotary evaporator for approximatelyone hour. Any solids formed are collected to provide the product byfiltration on a scintered glass funnel. The organic phase is washed withwater and then dried to constant weight in vacuo to provide the product.

DAla-D-⁶² Nal-Ala-Trp-D-Phe-Lys-NH₂, 35.

The hexapeptide Benzyloxycarbonyl-DAla-D-.sup.βNal-Ala-Trp-D-Phe-Lys-(Boc)-NH₂ (34, 1.02 mmol) is added to a roomtemperature solution of trifluoroacetic acid (30 mL), dimethylsulfide(14 mL), 1,2 ethanedithiol (7 mL) and anisole (2.2 mL) in methylenechloride (15 mL). The homogeneous reaction mixture is allowed to stirfor 15 minutes. After this period of time, anhydrous ether (450 mL) isadded to cause precipitation of the crude biologically active peptideproduct 35. This product is isolated by filtration on a scintered glassfunnel or by decantation. The resultant product is dissolved in waterand lyophilized. The lyophilized product can be further purified bymedium pressure chromatography on a 26×460 mm glass column containingLichroprep™ RP-18 column packing material (C-18, 25-40 nm, irregularmesh). After injection of the peptide as a solution in water, the columnis eluted at a flow rate of 9 mL per minute with a shallow gradient of 0to 25% methanol for 5-20 hours, and then by a gradient of 25 to 55%methanol over ca. 48 hours. The methanol concentration of the gradientis then increased at a rate of 2% per hour. During the elution, theremainder of the solvent composition is made up of water containing 0.2%trifluoroacetic acid. The product (35) is identified by HPLC and isisolated by concentration of the appropriate elution volumes.

The invention has been described in detail with particular reference topreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of this disclosure may makevariations and modifications within the spirit and scope of theinvention.

We claim:
 1. A peptide of the formulaA₁ -A₂ -Ala-Trp-DPhe-A₅, where A₁is Gly, DAla, βAla, Met, Pro, Sar, Ava, Aib, a N-lower alkylaminocarboxylic acid, a N,N-bis-lower alkyl amino-carboxylic acid or alower alkyl aminocarboxylic acid, wherein the lower alkyl groupcomprises 2 to about 6 straight-chain carbon atoms; A₂ is DTrp, D.sup.βNal, D-4-Y-Phe-or 5-Y-D-Trp, wherein Y is Cl, Br, F or H; A₅ is A₃ -A₄-A_(5'), A₃ -A₅, A₄ -A₅ or A₅, wherein(a) A₃ is Ala, Gly, DAla, Pro ordesAla; (b) A₄ is Ala, Gly, DAla, Pro, a linear lower alkylaminocarboxylic acid, or desAla; and (c) A_(5') is Lys(ε-R₁, R₂)-Z, Orn(δ-R₁,R₂)-Z, NH(CH₂)_(x) N(R₃,R₄), Lys-Z, Orn-Z or Arg-Z; wherein R₁ isa linear lower alkyl group or H atom; R₂ is a linear lower alkyl groupor H atom; but R₁ and R₂ cannot both be H; R₃ is a linear lower alkylgroup or H atom; R₄ is a linear lower alkyl group or H atom; Z isNH(linear lower alkyl group), N(linear lower alkyl group)₂, O-(linearlower alkyl group), NH₂ or OH, wherein the linear lower alkyl group isas defined as the lower alkyl group alkyl; x is 2 through 6; and organicor inorganic addition salts of the above.
 2. The peptide according toclaim 1, wherein A₁ is Gly, or DAla.
 3. A peptide of the formulaA₁ -A₂-Ala-Trp-DPhe-A₅, wherein A₁ is DAla; A₂ is DTrp, D.sup.β Nal,D-4-Y-Phe-or 5-Y-D-Trp, wherein Y is OH, Cl, Br, F or H; A₅ is A₃ -A₄-A5', A₃ -A₅ A₄ -A₅ or A₅, wherein(a) A₃ is Ala, Gly, DAla, Pro ordesAla; (b) A₄ is Ala, Gly, DAla, Pro, a linear lower alkylaminocarboxylic acid, or desAla; and (c) A_(5') is Lys(ε-R₁,R₂)-Z,Orn(δ-R₁,R₂)-Z, NH(CH₂)_(x) N(R₃,R₄), Lys-Z, Orn-Z or Arg-Z; wherein R₁is a linear lower alkyl group or H atom; R₂ is a linear lower alkylgroup or H atom; but R₁ and R₂ cannot both be H; R₃ is a linear loweralkyl group or H atom; R₄ is a linear lower alkyl group or H atom; Z isNH(linear lower alkyl group), N(linear lower alkyl group)₂ O-(linearlower alkyl group), NH₂ or OH, wherein the linear lower alkyl group isas defined as the lower alkyl group alkyl; x is 2 through about 6; andorganic or inorganic addition salts of the above.
 4. The peptideaccording to claim 1, wherein A₂ is DTrp or D.sup.β Nal.
 5. The peptideaccording to claim 1, wherein A₂ is D.sup.β Nal.
 6. The peptideaccording to claim 1, wherein A₅ is A₃ -A₄ -A_(5').
 7. The peptideaccording to claim 1, wherein A₅ is A₃ -A_(5').
 8. The peptide accordingto claim 1, wherein A₅ is A₄ -A_(5').
 9. The peptide according to claims1, 2, 3, 4 or 5 wherein A₅ is A_(5').
 10. A peptide having theformulaDAla-D.sup.β Nal-Ala-Trp-DPhe-LysNH₂, DAla-D.sup.βNal-Ala-Trp-DPhe-Lys(ξiPr)NH₂, DAla-DTrp-Ala-Trp-DPhe-Lys(ξiPr)NH₂,DAla-DTrp-Ala-Trp-DPhe-LysNH₂, DAla-D.sup.β Nal-Ala-Trp-DPhe-NH(CH₂)₅NH₂, or NH₂ (CH₂)₅ CO-D.sup.β Nal-Ala-Trp-DPhe-NH(CH₂)₅ NH₂ and organicor inorganic addition salts, thereof.
 11. A method of promoting therelease of growth hormone levels in an animal comprising administeringto the animal an effective amount of at least one of the peptides ofclaims 3 or
 10. 12. The method of claim 11, wherein the animal is amammal.
 13. The method of claim 11, wherein the animal is a human.
 14. Apharmaceutical composition for promoting the release of growth hormonelevels in animals comprising an effective mount of at least one of thepeptides of claims 1, 9, or 10 and a pharmaceutically acceptable carrieror diluent.
 15. A method of promoting the release and elevation of bloodgrowth hormone levels by administering the peptide of claim 1 in asynergistic amount with a second compound, wherein the second compoundis a compound which acts as an agonist at the growth hormone releasinghormone receptor or inhibits the effect of somatostatin.
 16. Thepharmaceutical composition of claim 14, which further comprises a secondcompound which acts as an agonist at the growth hormone releasinghormone receptor or inhibits the effects of somatostatin.
 17. A methodof promoting the release and elevation of blood growth hormone levels byadministering the peptide of claim 1 in a synergistic amount with atleast a Group 1 polypeptide or a Group 2 polypeptide, wherein the Group1 polypeptide is selected from any of the naturally occuring growthhormone releasing hormones and functional equivalents thereof and theGroup 2 polypeptide is selected from the group of polypeptidescomprising;Tyr-DArg-Phe-NH₂ ; Tyr-DAla-Phe-NH₂ ; Tyr-DArg (NO₂)-Phe-NH₂; Tyr-DMet (O)-Phe-NH₂ ; Tyr-DAla-Phe-Gly-NH₂ Tyr-DArg-Phe-Gly-NH₂ ;Tyr-DThr-Phe-Gly-NH₂ ; Phe-DArg-Phe-Gly-NH₂ ; Tyr-DArg-Phe-Sar-NH₂ ;Tyr-DAla-Gly-Phe-NH₂ ; Tyr-DArg-Gly-Trp-NH₂ ; Tyr-DArg(NO₂)-Phe-Gly-NH₂; Tyr-DMet (O)-Phe-Gly-NH₂ (NMe)Tyr-DArg-Phe-Sar-NH₂ ;Tyr-DArg-Phe-Gly-ol; Tyr-DArg-Gly-(NMe)Phe-NH₂ ; Tyr-DArg-Phe-Sar-olTyr-DAla-Phe-Sar-ol Tyr-DAla-Phe-Gly-Tyr-NH2;Tyr-DAla-(NMe)Phe-Gly-Met(O)-ol; Tyr-DArg-(NMe)Phe-Gly-Met(O)-ol;Gly-Tyr-DArg-Phe-Gly-NH₂ ; Tyr-DThr-Gly-Phe-Thz-NH₂ ;Gly-Tyr-DAla-Phe-Gly-NH₂ ; Tyr-DAla-Phe-Gly-ol;Tyr-DAla-Gly-(NMe)Phe-Gly-ol; Tyr-DArg-Phe-Sar-NH₂ ;Tyr-DAla-Phe-Sar-NH₂ ; Tyr-DAla-Gly-(NMe)Phe-NH₂ ;Sar-Tyr-DArg-Phe-Sar-NH₂ ; Tyr-DCys-Phe-Gly-DCys-NH₂ (cylic disulfide);Tyr-DCys-Phe-Gly-DCys-NH₂ (free dithiol); Tyr-DCys-Gly-Phe-DCys-NH₂(cyclic disulfide); Tyr-DCys-Gly-Phe-DCys-NH₂ (free dithiol);Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH₂ ; Tyr-DAla-Phe-Sar-Tyr-Pro-Ser-NH₂ ;Tyr-DAla-Phe-Sar-Phe-Pro-Ser-NH₂ ; Tyr-DAla-Phe-Gly-Tyr-Hyp-Ser-NH₂ ;Tyr-DAla-Phe-Sar-Tyr-Hyp-Ser-NH₂ ; Tyr-DAla-Phe-Sar-Phe-Hyp-Ser-NH₂ ;Tyr-DArg-Phe-Gly-Tyr-Hyp-Ser-NH₂ ; Tyr-DArg-Phe-Sar-Tyr-Pro-Ser-NH₂ ;Tyr-DArg-Phe-Sar-Tyr-Hyp-Ser-NH₂ ; and Tyr-DArg-Phe-Gly-Tyr-Pro-Ser-NH₂; and organic or inorganic addition salts, thereof.
 18. The method ofclaim 17, wherein one uses a Group 1 polypeptide and a Group 2polypeptide.
 19. The peptide according to claim 3, wherein A₂ is D.sup.βNal.
 20. The peptide according to claim 3, having the formulaDala-D.sup.β Nal-Ala-Trp-DPhe-Lys-Z.
 21. A peptide having the formulaDAla-D.sup.β Nal-Ala-Trp-DPhe-Lys-NH₂ and organic or inorganic additionsalts thereof.
 22. A method of promoting the release and elevation ofgrowth hormone levels in an animal comprising administering to theanimal an effective amount of a peptide having the formula DAla-D.sup.βNal-Ala-Trp-DPhe-Lys-Z, wherein Z is NH(linear C₂ -C₁₀ alkyl group),N(linear C₂ -C₁₀ alkyl group)₂, O-(linear C₂ -C₁₀ alkyl group), NH₂ orOH, and organic or inorganic addition salts thereof.
 23. The method ofclaim 22, wherein the peptide has the formula DAla-D.sup.βNal-Ala-Trp-DPhe-Lys-NH₂.
 24. A pharmaceutical composition comprising aneffective amount of a peptide having the formula DAla-D.sup.βNal-Ala-Trp-DPhe-Ly-Z and a pharmaceutically acceptable carrier ordiluent, wherein Z is NH(linear C₂ -C₁₀ alkyl group), N(linear C₂ -C₁₀alkyl group)₂, O-(linear C₂ -C₁₀ alkyl group), NH₂ or OH, and organic orinorganic addition salts thereof.
 25. The pharmaceutical composition ofclaim 24, wherein the peptide has the formula D-Ala-D.sup.βNal-Ala-Trp-DPhe-Lys-NH₂.
 26. A method of promoting the release andelevation of blood growth hormone levels by administering a peptidehaving the formula DAla-D.sup.β Nal-Ala-Trp-DPhe-Lys-Z wherein Z isNH(linear C₂ -C₁₀ alkyl group), N(linear C₂ -C₁₀ alkyl group)₂,O-(linear C₂ -C₁₀ alkyl group), NH₂ or OH, and organic or inorganicaddition salts thereof, in a synergistic amount with a second compound,wherein the second compound is a compound which acts as an agonist atthe growth hormone releasing hormone receptor or inhibits the effect ofsomatostatin.
 27. The method of claim 26, wherein the peptide has theformula DAla-D.sup.β Nal-Ala-Trp-DPhe-Lys-NH₂.
 28. A peptide of theformulaA₁ -A₂ -Ala-Trp-DPhe-A₅, where A₁ is Gly, DAla or βAla, A₂ isDTrp, D.sup.β Nal, D-4-Y-Phe-or 5-Y-D-Trp, wherein Y is OH, Cl, Br, F orH; A₅ is A₃ -A₄ -A_(5'), A₃ -A_(5'), A₄ -A₅, or A_(5'), wherein(a) A₃ isAla, Gly, DAla, or Pro; (b) A₄ is Ala, Gly, DAla, Pro, or a linear loweralkyl aminocarboxylic acid, and (c) A₅, is Lys(ε-R₁, R₂)-Z,Orn(δ-R₁,R₂)-Z, NH(CH₂)_(x) N(R₃,R₄), Lys-Z, Orn-Z or Arg-Z;wherein R₁is a linear lower alkyl group or H atom; R₂ is a linear lower alkylgroup or H atom; but when R₁ is H, R₂ is not H; and when R₂ is H, R₁ isnot H; R₃ is a linear lower alkyl group or H atom; R₄ is a linear loweralkyl group or H atom; Z is NH(linear lower alkyl group), N(linear loweralkyl group)₂, O-(linear lower alkyl group), NH₂ or OH, wherein thelinear lower alkyl group; x is 2 through 15; and organic or inorganicaddition salts of the above.
 29. The peptide according to claim 28,wherein A₂ is DTrp or D.sup.β Nal.
 30. A peptide of the formulaA₁-DβNal-Ala-Trp-DPhe-A₅, where A₁ is Gly, DAla or βAla; A₅ is A₃ -A₄-A_(5'), A₃ -A_(5'), A₄ -A₅, or A_(5'), wherein(a) A₃ is Ala, Gly, DAla,or Pro; (b) A₄ is Ala, Gly, DAla, Pro, or a linear lower alkylaminocarboxylic acid, and (c) A_(5') is Lys(ε-R₁, R₂)-Z, Orn(δ-R₁,R₂)-Z,NH(CH₂)-N(R₃,R₄), Lys-Z, Orn-Z or Arg-Z; wherein R₁ is a linear loweralkyl group or H atom; R₂ is a linear lower alkyl group or H atom; butwhen R₁ is H, R₂ is not H; and when R₂ is H, R₁ is not H; R₃ is a linearlower alkyl group or H atom; R₄ is a linear lower alkyl group or H atom;Z is NH(linear lower alkyl group), N(linear lower alkyl group)₂,O-(linear lower alkyl group), NH₂ or OH, wherein the linear lower alkylgroup x is 2 through 15; and organic or inorganic addition salts of theabove.
 31. A peptide of the formulaDAla-A₂ -Ala-Trp-DPhe-A₅, where A₂ isDTrp, D.sup.β Nal, D-4-Y-Phe-or 5-Y-D-Trp, wherein Y is OH, Cl, Br, F orH; A₅ is A₃ -A₄ -A_(5'), A₃ -A_(5'), A₄ -A₅, or A_(5'), wherein(a) A₃ isAla, Gly, DAla, or pro; (b) A₄ is Ala, Gly, DAla, Pro, or a linear loweralkyl aminocarboxylic acid, and(c) A₅, is Lys(ε-R₁, R₂)-Z,Orn(δ-R₁,R₂)-Z, NH(CH₂)_(x) N(R₃,R₄), Lys-Z, Orn-Z or Arg-Z; wherein R₁is a linear lower alkyl group or H atom; R₂ is a linear lower alkylgroup or H atom; but when R₁ is H, R₂ is not H; and when R₂ is H, R₁ isnot H; R₃ is a linear lower alkyl group or H atom; R₄ is a linear loweralkyl group or H atom; Z is NH(linear lower alkyl group), N(linear loweralkyl group)₂, O-(linear lower alkyl group), NH₂ or OH, wherein thelinear lower alkyl group is 2 through about 10 carbon atoms; x is 2through 15; and organic or inorganic addition salts of the above. 32.The peptide of claim 31, wherein A₂ is DβNal.
 33. A pharmaceuticalcomposition comprising an effective amount of the peptide of claim 28 ina pharmaceutically acceptable carrier or diluent.
 34. A peptide of theformulaDAla-A₂ -Ala-Trp-DPhe-A₅, wherein A₂ is DTrp or DβNal; A₅ is A₃-A₄ -A_(5'), A₃ -A_(5'), A₄ -A₅, or A_(5'), wherein(a) A₃ is Ala, Gly,DAla, or Pro; (b) A₄ is Ala, Gly, DAla, Pro, or a linear lower alkylaminocarboxylic acid, and (c) A_(5') is Lys(ε-R₁, R₂)-Z, Orn(δ-R₁,R₂)-Z,NH(CH₂)_(x) N(R₃,R₄), Lys-Z, Orn-Z or Arg-Z; wherein R₁ is a linearlower alkyl group or H atom; R₂ is a linear lower alkyl group or H atom;but when R₁ is H, R₂ is not H; and when R₂ is H, R₁ is not H; R₃ is alinear lower alkyl group or H atom; R₄ is a linear lower alkyl group orH atom; Z is NH(linear lower alkyl group), N(linear lower alkyl group)₂,O-(linear lower alkyl group), NH₂ or OH, wherein the linear lower alkylgroup; x is 2 through 15; and organic or inorganic addition salts of theabove.
 35. A pharmaceutical composition comprising an effective amountof the peptide of claim 34 in a pharmaceutically acceptable carrier ordiluent.